Objective To construct a prokaryotic expression system of norovirus (NoⅤ) G Ⅱ-4 strain P protein (P particle and P dimer) and to explore its binding activity and patterns with HBGAs receptor.Methods P domain sequence of GZ121 NoⅤ ORF2 gene was cloned and its phylogenic tree was constructed to identify the gene cluster.The pGEX-4T-1-based expression plasmids were constructed respectively by inserting P domain gene fragments with hinge and P-CDCRGDCFC without hinge,and then transformed into BL21 to express fusion proteins,which was induced with 0.6 mmol/L IPTG at 22℃ overnight.P proteins were purified by thrombin cutting and characterized by FPLC.The binding patterns of NoⅤ P protein to HBGAs antigens were analyzed by EIA.Results P region gene of GZ121 belonged to genotype G Ⅱ.4/2004 cluster.SDS-PAGE analysis showed the relative molecular weight of P particle and P dimer was about 36×103,which was consistent with other reports.The peak appeared at 830×103 confirmed the formation of P particle by FPLC.The expression of P protein was further confirmed by Western blot.The EIA results showed that GZ121 P protein could bind to saliva of A-group,B-group and O-group secretors,but not to nonsecretor.The binding affinity of P particle was 80-100 times higher than that of P dimer.Compared with VA387 P particle,it showed stronger binding affinity to O-group,but weaker to A-group.Conclusion The NoⅤ GⅡ-4 GZ121 P proteins including P particle and P dimer were successfully expressed and HBGAs receptor binding assays were established.This pave the way for further studies on the evolution dynamics of NoⅤ G Ⅱ.4 strains and the development of NoⅤ vaccines.

Objective To study the proportion and sonographic features of various kinds of diseases in the polypoid lesions of the gallbladder (PLG) diagnosed by ultrasonography, and to explore the reliability of ultrasonography for the diagnosis of PLG. Methods A total of 556 cases of PLG diagnosed by ultrasonography were classified basing on the results of pathological examination. And the accordance rate between ultrasonography and pathological examination in the PLG with different size was analyzed. Results Among the 556 cases of PLG, 446 (80.2%) had cholesterol polyps, 86 (15.5%) adenomatous polyps, 18 (3.2%) inflammatory polyps, and 6 (1.1%) myoadenoma. In 97.3% of the cholesterol polyps, the diameter was less than 1.0 cm, and 64.6% of the cases had multiple polyps. In the cholesterol polyps, the accordance rate between ultrasonography and pathological examination was 91.3%. Of the adenomatous polyps, 61.6% had a diameter over 1.0 cm, and 34.0% showed atypical hyperplasia or malignancy. However, 25% of the malignancies were found in the adenomatous polyps with a diameter less than 1.0 cm. Conclusions More than 80% of PLG are cholesterol polyps and 15.5% are adenomatous polyps. Most of the PLG with malignant transformation sized over 1.0 cm in diameter. However, the adenomatous polyps with a diameter ranged from 0.7 to 0.9 cm also have malignant tendency. In the cholesterol polyps, the accordance rate between ultrasonography and pathological examination is 91.3%, showing that ultrasonography is reliable for the diagnosis of the disease.

OBJECTIVE: To explore the significance of the combination of factor analysis and systematic cluster analysis in classification of traditional Chinese medical syndromes in patients with posthepatitic cirrhosis, and to provide a scientific basis for the criterion of the classification. METHODS: We designed a clinical questionnaire according to the clinical characteristics and the demands of traditional Chinese medical information collection for patients with posthepatitic cirrhosis. By means of clinical epidemiological research, with the four diagnosis methods for clinical information collection of traditional Chinese medicine, symptoms, physical signs, tongue conditions and pulse conditions in 310 patients with posthepatitic cirrhosis were collected, and the characteristics of traditional Chinese medical syndromes in these patients were explored with statistical methods, such as factor analysis, varimax and systematic cluster analysis. RESULTS: Analyzed by factor analysis and systematic cluster analysis with SPSS 11.0, the traditional Chinese medical syndromes in 287 of the 310 cases (92.58%) of posthepatitic cirrhosis could be classified. The syndromes could be divided into 7 categories, which were internal accumulation of damp-heat (55 cases), insufficiency of the spleen with overabundance of dampness (74 cases), accumulation of blood stasis plus deficiency of liver-yin and kidney-yin (73 cases), accumulation of blood stasis plus deficiency of both blood and qi (40 cases), deficiency of both blood and qi (16 cases), deficiency of yin and blood heat (6 cases) and stagnation of the liver-qi and deficiency of the spleen (23 cases). The traditional Chinese medical syndromes in the other 23 cases could not be classified. CONCLUSIONS: The clinical information collected with the four diagnostic methods of traditional Chinese medicine can be classified into different categories with the factor analysis and systematic cluster analysis. The factor analysis and systematic cluster analysis can reveal the characteristics and regularity of traditional Chinese medical syndromes in patients with posthepatitic cirrhosis in a way, and have value in researching the syndromes of traditional Chinese medicine.

OBJECTIVE:To prepare Yugan oral liquid and to observe its therapeutic effect on chronic hepatitis B METHODS:Yugan oral liquid was prepared of milkvetch root,thorowax root and magnolia vine fruit etc and the quality standard was established RESULTS:Yugan oral liquid was reasonable in both formulation and preparation process with a total effective rate of 89 8%,which was more effective than that in control group CONCLUSION:Yugan oral liquid is reliable in preparation technique,controllable in quality and effective in treatment of chronic hepatitis B

Objectives To study the effect of VIM in Enterovirus 71 (EV71) infection of (human brain microvascular endothelial cells (HBMEC) and elaborating the mechanism of EV71 infection in the nervous system. Methods Knocked out the VIM by CRISPR technology , the differences in EV71 absorption , replication , release between wild VIM and VIM knocked-out (VIM-KO) HBMEC were detected by fluorescence quantitative PCR. Results 4 ℃ absorption experiment conformed that EV71 adsorption in VIM- KO is 40% less than in the normal HBMEC. After EV71 infect HBMEC for 48 h (48 h p. i.), the quantitative PCR result showed intracellular viral RNA in VIM-KO was only 1/12 of that in the normal HBMEC. Also the extracellular viral RNA was quantified, and the number of cells in VIM-KO had been reduced 1.4 times compared with the normal HBMEC. Conclusions Once VIM knocking out, EV71 attachment has been obviously reduced. Meanwhile, the level of viral RNA replication and release are decreased compared with the normal HBMEC. VIM may be an attachment receptor of EV71 in HBMEC , when the virus invades HBMEC with the binding of VIM. Moreover , VIM plays an important role in the replication and release of EV71.

Objective: To investigate the sensitization of physiological (10~(-9)mol/L) and pharmacological (10~(-5)mol/L) concentrations of melatonin on cell line MCF-7 for adriamycin and its mechanism. Methods: (1) MTT was applied to test the changes in inhibition ratio and IC_(50) of call line MCF-7 for adriamycin before and af-ter incubation with melatonin. (2) Flow cytometry was used to observe the effect of different concentrations of melatonin, adriamycin and melatonin plus adriamycin on cell apoptosis. (3) Western blot was employed to de-termine the expression of p53 and bcl-2 in MCF-7 cells incubated with melatonin, adriamycin and melatonin plus adriamycin. Results: (1) MTT method showed that adriamycin had inhibitive effect on the growth of MCF-7 cells in a dose- and time-dependent manner. The IC_(50) of cell line MCF-7 for adnamycin before treat-ment with melatonin was 0.62±0.07ug/mL (P>0.05). The IC50 of cell line MCF-7 for adriamycin incubated with physiological and pharmacological concentrations of melatonin was 0.59±0.09ug/mL and 0.42±0.02ug/mL, re-spectively, with a significant difference (P<0.01). (2) Flow cytometry method showed that adriamycin could promote apoptosis of MCF-7, and no changes in the apoptosis index were observed as the concentration of melatonin was changed (P>0.05). With the same concentration of adriamycin, the apoptosis index of cells treated with physiological concentration of melatonin plus adriamycin was not changed (P=>0.05), but the apop-tosis index of cells treated with pharmacological concentrations of melatonin plus addamycin was increased significantly. The concentration of adriamycin had no effect on the apoptosis index. (3) Western blot showed that P53 protein was expressed at a lower level and bcl-2 protein was highly expressed. Physiological concen-trations of melatonin increased the expression of p53 and decreased bcl-2 expression in a dose - dependent manner. The concentration of addamycin had no effect on the expression of p53 and bcl-2 proteins. Conclu-sion: (1) Physiological concentrations of melatonin had no effect on the anti-cancer effect of adriamycin. Phar-macological concentrations of melatonin showed sensitization of MCF-7 cells for adriamycin. (2) With a lower concentration of adriamycin, the promotion of apoptosis may be part of the mechanism of sensitization effect of melatonin. With the increase of adriamycin concentration, the cytotoxic mechanism of melotonin became more and more important. (3) Physiological concentration could increase the expression of p53 and decrease bcl-2 expression in ER~+ breast carcinoma cell line MCF-7 in a dose -dependent manner. The apoptosis involv-ing p53 and bcl-2 passway was part of the mechanism of sensitization effect of melatonin for addamycin.

Objective To test the inhabiting effect of Lactobacillus acidophilus on E.coli O157: H7 in intestinal colonization and explore its mechanism. Methods The suppressive effects of Lactobacillus acidophilus against E.coli O157:H7 adhering to Ht29 cells were carried out by competition , exclusion and replacement as-says. Furthermore, we evaluated the cytokine levels of IL-4, IL-12, and INF-γ in serum of mice. In addition, E.coli O157:H7 fecal shedding was monitored and the pathological changes of intestines were observed in mice. Results The competition, exclusion and replacement assays showed Lactobacillus acidophilus inhibited E.coli O157:H7 adhering to Ht29 cells. In vivo, the mice of treatment group were induced significantly higher level of IL-4, IL-12, and INF-γ, though prevention group induced IL-12 only. Fifteen days after E.coli O157:H7 infec-tion, there were 8 mice (80%) in prevention group and 5 mice (50%) in treatment group stopped shedding. Moreover, the pathological changes of intestines of both prevention group and treatment group appeared normal , but control groups showed seriously damaged in intestinal villus. Conclusion Lactobacillus acidophilus inhibits E.coli O157:H7 in intestinal colonization and the preventative effect was better than treatment effect. Thus , Lac-tobacillus acidophilus can be used for E.coli O157:H7 in prevention and treatment infection as probiotics.

ObjectiveTo investigate the effect of arsenic trioxide (As2O3) on the metastasis capability of Ewing's sarcoma ceils. MethodsMTT assays were performed to choose appropriate concentrations of As2O3 (＜ 2 μmol/L) for the experiments.Migration and invasion assays were performed to assess the effect of As2O3 on the metastasis of Ewing's sarcoma cells. Changes in matrix metalloproteinase(MMP)-9 expressions were detected by gel zymography assay and the phosphoinositide 3-kinase/AKT(PI3K-AKT)pathway was investigated using Western blot. ResultsThe amount of Ewing's sarcoma cells across basal membrane of Transwell in migration and invasion assay decreased gradually with the increase in As2O3 concentration. The average quantities of A-673 across the membrane after treatment by gradual concentrations accounted for 54.3 ％,49.0 ％ and 17.0 ％ of that of untreated group respectively in migration assay (F=112.78,P ＜ 0.01), while 52.7 ％, 32.3 ％ and 10.3 ％ in invasion assay(F =183.76, P ＜ 0.01). Similarly, the percentage of RD-ES was 46.0 ％,39.0 ％ and 8.0 ％ in migration assay (F =408.25,P ＜ 0.01) and 58.7 ％,22.3 ％ and 9.0 ％ in invasion assay (F =373.25, P ＜ 0.01)respectively. The difference had statistics significance.The expression of MMP-9 was suppressed by As2O3 treatment according to gel zymography assay.Western blot assay showed that PI3K-AKT pathway was inhibited and nuclear factor kappa B(NF-κB)was inactivated.ConclusionLow-concentration As2O3 may inhibit metastasis capability of Ewing's sarcoma cells.

Objective To investigate the clinical application value of ultrasonic rapid paraffin section in intraoperative diagnosis of breast tumor.Methods Twenty-five patients of breast tumor specimens were admitted from December 2012 to December 2013,including 19 cases from surgery,4 cases from gynecology,2 cases from medical patients.Twenty-five cases were used with ultrasonic rapid paraffin section and the remaining tissues were used as conventional paraffin section,then compared the two methods.Results This study took two different ways but the same results were achieved with 23 cases,the diagnostic accuracy was 92.0％ (23/25).Two of 25 cases were misdiagnosed with the misdiagnosis rate was 8.0％ (2/25).One patient appeared delayed diagnosis,accounting for 4.0％(1/25).After 25 cases of breast cancer biopsy treated with ultrasonic rapid paraffin section,benign lesions was 20 patients,with the highest incidence of breast adenosis of 9 cases (36.0％,9/25).The malignant lesions was 4 patients with invasive ductal carcinoma in which the highest incidence,accounting for 3 cases (12.0％,3/25).Delay diagnosed 1 case of intraductal papilloma.Conclusion Ultrasonic rapid parafifin section is widely used in clinical application,with high quality and diagnostic biopsy rate and other characteristics,so it is worthy of clinical application.

Objective To study the factors influencing epidemiological characteristics and virulence of Enterovirus 71 (EV71) in Guangdong province from 2008 to 2010. Methods RNA was extracted from collected samples or cultured virus , then reversing transcription into cDNA. We amplified full-length EV71-VP1 using poly-merase chain reaction technology , then conducted sequence alignment and established phylogenetic tree with MEGA software (version 5.0) to confirm the genotype of EV71. The association between severity of clinical symp-toms and sex, age, viral genotype and VP1 variation was also analyzed using Logistic regression. Results The genotype of the predominant epidemical strain was C4a in Guangdong from 2008 to 2010. However , this subtype had already differentiated into 4 subgroups (C4a1- C4a4). There was no correlation between clinical syndrome and sex or viral genotype; the severity of symptoms was negatively correlated with age: before 4 years old, varia-tion A289T can easily lead to severe cases, increasing the risk of infection (P<0.05, OR = 2.360, 95%CI:1.163~ 4.659). Conclusion The main epidemical EV71 strain is C4a1 in Guangdong province. The emerging differen-tiation and simultaneous prevalence should merit attention to strengthen relevant surveillance; and the protection of the susceptible population should be reinforced during EV71 prevalence.